Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development

Mfn2 is a mitochondrial fusion protein with bioenergetic functionsimplicated in the pathophysiology of neuronal and metabolicdisorders. Understanding the bioenergetic mechanism of Mfn2may aid in designing therapeutic approaches for these disorders.Here we show using endoplasmic reticulum (ER) or mitochondria-targeted Mfn2 that Mfn2 stimulation of the mitochondrial meta-bolism requires its localization in the ER, which is independent ofits fusion function. ER-located Mfn2 interacts with mitochondrialMfn1/2 to tether the ER and mitochondria together, allowing Ca2+transfer from the ER to mitochondria to enhance mitochondrialbioenergetics. The physiological relevance of these findings isshown during neurite outgrowth, when there is an increase inMfn2-dependent ER-mitochondria contact that is necessary forcorrect neuronal arbor growth. Reduced neuritic growth in Mfn2KO neurons is recovered by the expression of ER-targeted Mfn2 oran artificial ER-mitochondria tether, indicating that manipulationof ER-mitochondria contacts could be used to treat pathologicconditions involving Mfn2

Estudio de los peroxisomas sobre la función mitocondrial

Curs 2018-2019En este Trabajo de Fin de Grado se presentan los resultados del estudio de los peroxisomas y de su importancia para el correcto funcionamiento de las mitocondrias. En un estudio anterior de microarray se mostró que la estimulación sináptica neuronal de neuronas corticales de ratón provocaba un aumento en los niveles de peroxin 5 (Pex5p). Pex5p es una proteína de importación de proteínas al peroxisoma que podría jugar un papel crítico en la regulación de la autofagia. Disfunciones en la autofagia contribuyen al desarrollo y progresión de numerosas enfermedades humanas, como el cáncer, los trastornos neurodegenerativos, el envejecimiento y las enfermedades metabólicas. Por otro lado, los orgánulos no están aislados en la célula, sino que, interactúan entre ellos regulando la actividad celular. Por lo que, la pérdida de genes peroxisomales Pex da lugar a anomalías en la estructura mitocondrial y su función metabólica, generando problemas en la actividad celular. Así pues, el objetivo principal del proyecto es estudiar el efecto de los peroxisomas sobre la función mitocondrial y su interacción con las mitocondrias. Para ello, mediante Westerns blot, se ha determinado la participación de la proteína Pex5p a las 24 horas de la estimulación sináptica neuronal con bicuculline y, posteriormente, se han estudiado los efectos que provoca la expresión de Pex5p en las mitocondrias de la línea celular de células del riñón de rata (NRK) y, paralelamente, en neuronas corticales de rata estimuladas sinápticamente durante 24 horas. (Se utiliza la línea celular NRK porque son células fáciles de transfectar y estudiar, a diferencia de las neuronas, y en el corto tiempo disponible para realizar el trabajo era la opción más favorable.) También se ha estudiado el papel de la mitofusina 2 en las interacciones de los peroxisomas con las mitocondrias. Ya que recientemente se ha propuesto que la mitofusina 2, una proteína de membrana mitocondrial que permite interacciones entre el retículo endoplasmático y las mitocondrias, puede estar implicada en las conexiones peroxisoma-mitocondria. Los resultados del proyecto muestran que se producen anomalías mitocondriales en células NRK cuando se reduce la expresión de Pex5p y que la mitofusina 2 parece estar implicada en las interacciones peroxisoma-mitocondria. Estos resultados sirven de punto de partida para futuros experimentos en que se estudie el papel de Pex5p en la neuroprotección y se confirme que la mitofusina 2 participa en las interacciones peroxisoma-mitocondria.This Final Degree Project exposes the study of peroxisomes for the correct operation in mitochondrial function. A microarray analysis shows an increase of peroxin 5 (Pex5p) levels in mouse cortical neurons stimulated synaptically. Pex5p is a peroxisomal import protein that could play a critical role in the regulation of autophagy. Autophagy dysfunctions contribute to development and progression of numerous human diseases, such as cancer, neurodegenerative disorders, aging and metabolic diseases. Furthermore, organelles interact with each other regulating cell activity. So, the loss of peroxisomal Pex genes leads to problems in cellular activity by abnormalities in the mitochondrial structure. Therefore, the main objective of the project is to study the effect of peroxisomes on mitochondrial function and its interaction with them. The participation of the Pex5p protein is determined by Westerns blot of neuronal cells stimulated 24 hours with bicuculline. Then, the mitochondrial effect caused by the expression of Pex5p in natural kidney cells (NRK) and, in parallel, in rat cortical neurons stimulated synaptically for 24 hours are studied. (The NRK cell line is used because they are cells easy to transfect and study, unlike neurons, and in the short time available to perform the work was the most favourable option.) The role of mitofusin 2 has also been studied in the mitochondria-peroxisome interactions. Such it has been proposed recently that the mitochondrial membrane protein mitofusin 2, which allows interactions between the endoplasmic reticulum and the mitochondria, may participate in the peroxisome-mitochondrial connections. The results of the project show mitochondrial abnormalities in NRK cells when Pex5p expression is reduced and a possible peroxisome-mitochondrion interaction by mitofusin 2. These results serve as starting point for future studies on the role of Pex5p in neuroprotection and in peroxisome-mitochondrion interactions by mitofusin 2

Synaptic Activity Regulates Mitochondrial Iron Metabolism to Enhance Neuronal Bioenergetics

Synaptic activity is the main energy-consuming process in the central nervous system. We are beginning to understand how energy is supplied and used during synaptic activity by neurons. However, the long-term metabolic adaptations associated with a previous episode of synaptic activity are not well understood. Herein, we show that an episode of synaptic activity increases mitochondrial bioenergetics beyond the duration of the synaptic activity by transcriptionally inducing the expression of iron metabolism genes with the consequent enhancement of cellular and mitochondrial iron uptake. Iron is a necessary component of the electron transport chain complexes, and its chelation or knockdown of mitochondrial iron transporter Mfrn1 blocks the activity-mediated bioenergetics boost. We found that Mfrn1 expression is regulated by the well-known regulator of synaptic plasticity CREB, suggesting the coordinated expression of synaptic plasticity programs with those required to meet the associated increase in energetic demands